The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. SV40 DNA replication in infected cells and in cell-free reactions has served as a model system for eukaryotic DNA replication, facilitating the identification and characterization of 10 human proteins that, together with the viral T antigen, are necessary and sufficient to reconstitute SV40 DNA replication in vitro. The same cellular proteins are required for host cell DNA replication, suggesting that some mechanisms used for host DNA replication resemble those elucidated in the viral system. However, the early steps of SV40 DNA replication: recognition of the origin of DNA replication, recruitment of replication enzymes to the origin, and DNA helicase activity during unwinding of parental DNA at the replication forks are all performed by T antigen. Multiple cellular proteins are required to perform these early steps in mammalian DNA replication, as well as to regulate them in response to replication stress. The proposed research program is designed to increase our understanding of the differences and similarities between viral and host cell DNA replication. Specific Aim 1 will combine genetics, biochemistry, and structural biology to investigate the mechanism by which SV40 T antigen interacts with Replication Protein A to mediate primer synthesis by DNA polymerase a-primase. In Specific Aim 2, we will continue to explore the structure and function of DNA polymerase a-primase, its interaction with T antigen, and its response to replication stress. In Specific Aim 3, our goal is to determine the function(s) of HDHB, a novel human DNA helicase that shares some properties with T antigen. The association of HDHB with Mre11/Nbs1/Rad50 and other unknown proteins will be studied using biochemical, genetic, and cell biological approaches, in the presence and absence of T antigen. These studies will provide a deeper understanding of how SV40 T antigen co-opts host replication proteins and may uncover novel mechanisms by which these helicases affect host genomic integrity. [unreadable] [unreadable] [unreadable]